PROJECT SUMMARY/ABSTRACT Our long-term goal is to develop small-molecule therapies for heart failure (HF) that target the cardiac sarcoplasmic reticulum calcium-ATPase (SERCA). SERCA plays an essential role in normal cardiac function, clearing cytosolic calcium needed to relax muscle cells in each heart beat (diastole). A key molecular dysfunction in HF usually involves insufficient SERCA expression, leading to SERCA inactivation and impaired calcium transport in the cardiomyocyte. SERCA is now widely recognized as a therapeutic target, and its activation results in improved cardiac function in HF models. Therefore, we propose to develop small-molecule SERCA activators directed at the myocyte as an effective therapeutic approach for restoring normal function in the failing heart. This is innovative because discovery of pharmacologically viable SERCA activators would represent a major breakthrough in therapies for heart failure, as it deviates from known current therapeutic options. We recently discovered and validated HF600, a high-quality hit that activates SERCA and stimulates intracellular calcium transport in human iPSC cardiomyocytes. We now intensify our collaboration and propose a flexible, fast and cost-effective drug development strategy beyond the typical discovery of hit molecules to generate novel pharmacologically viable molecules. Our central hypothesis is that hit-to-lead optimization around HF600 will produce novel therapeutic candidates for the pharmacological treatment of HF. Three specific aims will be pursued in this project to test this hypothesis: (1) design in the computer and synthesize series of pharmacologically viable SERCA activators built around the hit molecule HF600; (2) evaluate the functional activity of SERCA activators in situ, and in both human iPSC cardiomyocytes and animal-derived adult ventricular myocytes; and (3) determine therapeutic efficacy, safety and pharmacokinetics of SERCA activators. Therapeutic efficacy of small-molecule candidates will be tested using diseased iPSC cardiomyocytes (patient- derived and induced); SERCA activators will be evaluated through iPSC safety screening and pharmacokinetics studies. We now have extensive preliminary data showing that a new SERCA activator we built around HF600 reverses calcium mishandling in the diseased cardiomyocyte, and also protects it against arrhythmia with no apparent long-term cardiotoxicity. This excellent preliminary data provides mechanistic proof-of-principle for activating SERCA for HF therapy. Our outstanding multidisciplinary team and highly complementary approaches on a validated pharmacological target ensure successful discovery of novel candidates for the pharmacological treatment of patients with heart failure.